RECONSTITUTION OF DNA-END REPAIR PATHWAY IN PROKARYOTES

Abstract

Suggested is a method for engineering and/or editing the genome of prokaryotes encompassing the following steps: (i) providing a culture of prokaryotic cells, (ii) preparing a vector comprising an expression system encompassing at least one programmable DNA-binding and -cleaving protein, (iii) introducing said vector into said prokaryotic cells to target a specific DNA sequence in the genome of said prokaryotic cells.

Description

FIELD OF INVENTION

[0001] The present invention relates to genome engineering and editing in prokaryotes, particularly targeted modification of a prokaryotic genome, such as disruption of gene function (knock-out), deletion of genomic locus or insertion of DNA elements that may use vector systems to reconstitute DNA-end repair system in prokaryotes in combination with programmable nucleases.

STATE OF THE ART

[0002] Targeted genome engineering and editing relies on the capability to introduce precise DNA-cleavage at the genomic locus of interest and on the capability of the host cell to repair the cleavage site. Several programmable DNA-binding and -cleaving proteins have been developed that allow a precise introduction of double-strand DNA breaks (DSBs) at a specific genomic locus of interest in order to modify the DNA sequence flanking the cleavage site. Examples of such programmable DNA-cutting enzymes include Zn-finger or TAL nucleases, meganucleases and CRISPR-Cas9 [1, 2]. In eukaryotes DSBs are repaired by either endogenous non-homologous end-joining (NHEJ) or homologous recombination (HR) pathway. In the NHEJ pathway the DNA-breaks are enzymatically sealed by a set of proteins including the DNA-end binding protein Ku that recruits ligases to the cleavage site. Heterodimeric Ku protein specifically binds to the DNA-ends and mediates the repair of DSBs by promoting the formation of DNA-end synapsis and recruitment of recombination proteins, including DNA ligases. NHEJ repair is intrinsically erroneous and leads to deletion or insertion of few bases. These indel (insertion-deletion) mutations can cause frameshift mutation and thus to knockout protein encoding genes when the repair site is located within an open-reading-frame (ORF) [2]. Thus, a simple way to knock-out a gene of interest is to introduce DSB within its ORF using programmable DNA-cutting protein in order to induce the error-prone NHEJ pathway.

[0003] Due to the lack of NHEJ repair proteins in most prokaryotes, DSBs have to be repaired by homologous repair pathway, which requires the presence of a donor-template DNA that contains homologous sequences flanking the DSBs [3-5]. Otherwise, DSBs introduced in the genomic DNA (self-targeting) causes death of the prokaryotic host [3]. Therefore, the use of the DNA-cutting enzymes, like Cas9, meganucleases, TAL nucleases, Zn finger proteins for targeted gene modification in prokaryotes is coupled to the homologous recombination system and requires providing of homologous recombination template for each targeted DNA site. This limits the applicability of especially versatile CRISPR-based methods, such as multiplex genome editing and genome-wide knock-out (GeCKO) screenings [6]. As a matter of fact CRISPR-Cas9 technology is today's most promising tool for genome engineering, providing

[0004] the ability to perform multiplex gene modifications in a single cell at the same time and

[0005] the application of CRISPR-RNA libraries for genome-wide "loss-of-function" screening assays, both applicable in many eukaryotic model organisms.

[0006] Both methods cannot be transferred to prokaryotes due to the lack of NHEJ pathway. A method that reconstitutes the DNA-end repair pathway in prokaryotes would allow the adaptability of Cas9-based genome engineering/editing and genome-wide screening assays in prokaryotes.

[0007] Therefore, there is urgent need to develop vector systems and methods that reconstitutes the DNA-end repair pathway in prokaryotic host cells, which may allow the application of genome engineering and editing technologies in prokaryotic organisms.

[0008] The object of the present invention has been to overcome this limitation in prokaryotes by utilization of NHEJ and NHEJ-like repair pathways in order to reconstitute DNA-end repair system in prokaryotes

[0009] to prevent cell death caused by DSBs and

[0010] to enable the (erroneous) repair of DSBs created by programmable DNA-cleaving proteins like CRISPR-Cas9.

DESCRIPTION OF THE INVENTION

[0011] Object of the present invention is a method for engineering and/or editing the genome of prokaryotes (bacteria or archaea) encompassing the following steps:

[0012] (i) providing a culture of prokaryotic cells,

[0013] (ii) preparing a vector comprising an expression system encompassing at least one programmable DNA-binding and cleaving protein,

[0014] (iii) introducing said vector into said prokaryotic cells to target a specific DNA sequence in the genome of said prokaryotic cells.

[0015] More particularly, the method encompasses the following steps:

[0016] (i) providing a culture of prokaryotic cells,

[0017] (ii) preparing a vector comprising an expression cassette for a DNA-double strand break repair system encompassing

[0018] (a) at least one protein binding to the DNA-ends,

[0019] (b) at least one protein with DNA-ligase activity

[0020] (iii) introducing said vector into said prokaryotic cells to enable introduction of double strand DNA breaks according to claim 1 in the genome of said prokaryotic cells.

[0021] Even more preferred is the embodiment according to which the method encompasses the following steps:

[0022] (i) providing a culture of prokaryotic cells,

[0023] (ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 50 nucleotides (nt) guide sequence of said sgRNA being complementary to desired stretches within the non-coding and/or putative regulatory regions upstream of the translation start codon of at least one gene of said prokaryotic cell;

[0024] (iii) preparing a vector comprising an expression cassette encompassing

[0025] (a) at least one programmable DNA-binding and cleaving protein,

[0026] (b) at least one optionally modified sgRNA; and

[0027] (c) at least one DNA-end binding protein; and

[0028] (iv) transforming said culture of prokaryotic cells with said vectors by standard methods (e.g. chemical transformation, electroporation, conjugation or transduction) to target the genome for the presence of a DNA sequence that is complementary to the 10 to 50 nt guide sequence of said sgRNA or protein-based nucleases like TAL- or Zn-finger proteins.

[0029] In this context reference is made to the paper by T. Su et al. titled "A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome" (www.nature.com/Scientific Reports 6:37895/D01:10.1038/srep37895), published Nov. 24, 2016, which means after the priority date the present application is assigned to. The authors describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial genes in a homologous recombination-independent manner and without the use of selective markers. According to this study CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step without the prerequisite of a homologous DNA template. Obviously, the paper refers to the same problem and provides a similar solution, thus providing additional proof that the proposed technical teaching is effective.

BRIEF DESCRIPTION OF THE INVENTION

[0030] For transforming a culture of prokaryotic cells with plasmid vectors standard methods which are known in the art can be applied, such as chemical transformation, electroporation, conjugation or transduction. The vector can be a plasmid, a bacteriophage, a phagemid or a virus.

[0031] Few prokaryotic species contain genes that show functional similarity to the eukaryotic proteins involved in the NHEJ pathway. A well-known example is the NHEJ-like pathway in Mycobacterium tuberculosis mediated by the two proteins Ku (MtKu) and LigD (MtLigD). In order to proof our intention to induce the repair of DNA-ends in prokaryotes when using programmable DNA-cutting enzymes we analyzed the effect of MtKu and MtLigD on the toxicity of self-targeting Cas9-sgRNA ribonucleoproteins in Azotobacter vinelandii, Escherichia coli and Pseudomonas putida. To this aim, we designed two vectors, one that encodes the Cas9 protein (pB5-Para-Cas9-PsacB-sgRNA, FIG. 1A) and another vector that encodes Cas9, MtLigD and MtKu proteins (pB5-CLK_PsacB-sgRNA, FIG. 1B). Both vectors also comprise the expression cassette for the transcription of a sgRNA from the promoter PsacB. Using the restriction enzyme BbsI, we are able to modify the first 20 nucleotides of the sgRNA on both vectors, which determine the cleavage site by the Cas9 protein.

[0032] To solve the problem underlying the present invention a guide sequence into the vectors pB5-Para-Cas9-PsacB-sgRNA and pB5-CLK_PsacB-sgRNA was inserted that directs the Cas9 nuclease to the upp gene of A. vinelandii[7]. Since the upp gene is not essential, a toxicity of upp targeting Cas9 would be an indication for the detrimental effect of DSBs on cell viability per se. Indeed, the expression of upp-targeting Cas9-sgRNA complexes from the pB5-Para-Cas9-PsacB-sgRNA vector results in almost complete lack of viable A. vinelandii (compare FIGS. 2A and 2B). To proof the Cas9-specificity of the observed toxic effect two amino acids of Cas9, known to inactivate the nuclease activity of Cas9 (dCas9, D10A and H840A) were replaced. As can be seen in FIG. 2C the expression of dCas9-upp-sgRNA has no effect on the viability confirming that the toxicity is based on the DSB induced by wild-type Cas9 loaded with upp-targeting sgRNA. Next, the repair proteins MtKu and MtLigD with the self-targeting wildtype Cas9-upp-sgRNA were co-expressed. The results show clearly that in the presence of MtKu and MtLigD the number of the survived cells is significantly increased (compare FIG. 2B and FIG. 2D), indicating a promoting effect of MtKu and MtLigD on the repair of Cas9-induced DNA-cleavage in A. vinelandii. In eukaryotes up to 10% of the repair events by the NHEJ pathway results in mutation at the repair site. In order to test whether the repair with MtKu-MtLigD in A. vinelandii results in mutations at the targeted region, the viable clones on agar plates supplemented with 5-Fluorouracile (5-FU) were selected, whose presence in the growth medium is only toxic for the cells that contain an intact upp gene. Indeed, it was possible to isolate 5-FU resistant clones that were transformed with pB5-CLK_PsacB-sgRNA_upp55 vector containing a single guide sequence targeting the upp gene. Genomic DNA from 5-FU-resistent clones was prepared and a region spanning the upp gene was amplified by PCR and analyzed by Sanger sequencing.

[0033] As shown in FIG. 3, clones, which escaped the toxicity of Cas9-induced DSB at the upp gene, contain a large deletion 3-bp immediately upstream of the protospacer adjacent motif (PAM) 5''-NGG-3''. Cas9-sgRNA complexes are known to introduce DSB precisely within the target region 3''-upstream of the PAM. Therefore, the sequencing results strongly suggest that the upp gene was cleaved at the expected site by Cas9 nuclease followed by exonucleolytic degradation and sealing of the resulting DNA-ends.

[0034] In order to show the adaptability of our system to other prokaryotic species, the experiments were repeated in Pseudomonas putida (DSM12264). Conjugation of P. putida with the vector pB5-Cas9 that targets the genomic upp gene and plating of the conjugants on selective agar plates resulted in complete lack of viable colonies (FIG. 4A). However, co-expression of MtKu and MtLigD with Cas9 and upp-targeting sgRNA resulted again in the formation of viable clones, demonstrating a reduced toxicity of Cas9-induced genomic DNA breaks in the presence of MtKu and MtLigD.

[0035] Next, similar analyses in E. coli MG1655 were performed using spacer sequences that direct Cas9 to the genomic lacZ gene. As shown in FIG. 5, the co-induction of the repair proteins MtKu and MtLigD also reduced the toxicity of self-targeting Cas9-sgRNA complexes in E. coli, indicating a wide-range applicability of our system in different prokaryotic species. Thus, based on these results, one can conclude that the Cas9 technology and other programmable nucleases that introduce DSBs can be applied in prokaryotes by coupling the nuclease activity with repair proteins that reconstitute DNA-end repair pathway.

[0036] In order to test a nuclease-mediated introduction of mutations into the lacZ gene E. coli MG1655 was transformed either with the plasmid pB5-Para-Cas9-Pveg-LigD_Ku (FIG. 1C) that encodes for ParaBAD-driven Cas9, Pveg-driven LigD-Ku or with pB5-Para-Cas9-PvegLigD_Psac_Ku that encodes for ParaBAD-driven Cas9, Pveg-driven LigD and PsacB-driven Ku proteins. The cleavage of the lacZ gene was induced through a second transformation step by electroporation of the plasmid pUCP-PsacB-sgRNA-bgaI (FIG. 1E) containing the lacZ-targeting sgRNA transcription unit. The transformants were plated onto agar plates supplemented with ampicillin (100 .mu.g/ml), kanamycin (25 .mu.g/ml), arabinose (0.2% w/v) and X-Gal (80 .mu.g/ml) (one example is shown in FIG. 6). Up to 24 white colonies were picked and used for single-colony PCR amplification and Sanger sequencing of the lacZ region (forward primer: 5''-GATACGACGATACCGAAGACA-3'; reverse primer: 5''-GATAACTGCCGTCACTCCAG-3'). Sequencing results of five of such clones are shown in FIG. 7, demonstrating the deletion of eight (Clone3_SeqID_41 HA16) up to 243 base-pairs (Clone 1_SeqID_41 HA14) around the Cas9-cleavage site (sgRNA target region is indicated in blue, protospacer-adjacent motif in red). The present invention enables to introduce mutations into E. coli genome without the need of homologous recombination template, which is otherwise essential to modify the E. coli genome with CRISPR-Cas9 technology [3].

Further Objects of the Invention

[0037] Typically, the prokaryotic cells belong to bacteria or archaea, preferably bacteria.

[0038] The preferred vector is a plasmid or phage-DNA, which is usually introduced into the prokaryotic cell by means of transformation, transduction or conjugation

[0039] The programmable DNA-binding and cleaving proteins are preferably selected from the group consisting of Zn-finger, TAL nucleases, meganucleases and RNA-dependent CRISPR-associated nucleases, and more preferably from the group of CRISPR-Cas proteins belonging to class 2-type II CRISPR systems.

[0040] The most preferred programmable DNA-binding and cleaving proteins are Cas9 or Cpf1.

[0041] On the other side, the preferred DNA-end repair proteins are selected from the group consisting of proteins showing at least 30% identity in their primary sequence to protein Ku, and/or LigD of prokaryotes. The most preferred embodiment refers to DNA-end repair proteins which are selected from the group consisting of proteins Ku and/or LigD encoded by Gram-positive bacteria, more preferred encoded by Mycobacteria and particularly encoded by Mycobacterium tuberculosis.

[0042] Another object of the present invention refers to an expression system comprising

[0043] (a) at least one Cas9, modified Cas9 or Cpf1 protein,

[0044] (b) at least one optionally modified sgRNA or crRNA; and

[0045] (c) protein Ku and/or LigD.

[0046] Finally, other objects of the present invention cover:

[0047] (I) A vector comprising or consisting of the expression system as explained above.

[0048] (II) A process for gene knock-out, gene or single base pair deletion, replacement, editing, and/or for genome wide knock-out screening in a prokaryote encompassing the methods explained above.

[0049] (III) The use of DNA-end binding and -repair proteins in a process for genome engineering and editing in prokaryotes, particularly targeted modification of a prokaryotic genome, such as disruption of gene function (knock-out), deletion of genomic loci or insertion of DNA elements in prokaryotes in combination with programmable nucleases that work via introduction of DNA-double strand breaks.

CITED REFERENCES

[0049]

[0050] [1] HU J H et al. "Chemical Biology Approaches to Genome Editing: Understanding, Controlling and Delivering Programmable Nucleases", Cell Chem. Biol. 23: 47-73 (2016)

[0051] [2] HSU P D et al. "Development of CRISPR-Cas9 for genome engineering", Cell 157:1262-78 (2014)

[0052] [3] JIANG W et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas9 systems" Nat. Biotechnol 31:233-9 (2014)

[0053] [4] LI Y et al. "Metabolic engineering of E. coli genome via the CRISPR-Cas9 mediated genome editing" Metab. Engin. 31:13-21 (2015)

[0054] [5] SHALEM 0 ET AL. "Genome-scale CRISPR Cas-9 knockout screening in human cells" SCIENCE 343: 84-7 (2014)

[0055] [6] SETUBAL J C et al. "Genome sequence of Azotobacter vinelandii, an obligate aerobe spcialized to support diverse anaerobic metabolic processes" J. Bacteriol. 191: 4534-45 (2009)

EXAMPLES

Example 1

[0056] As shown in FIG. 2 the presence of Ku-LigD promotes the repair of DSB induced by Cas9 loaded with self-targeting sgRNA in A. vinelandii. Isolation of clones and sequencing of the targeted region showed a specific Cas9-induced DNA a break 3-nt upstream of the PAM sequence, exonucleolytic degradation and ligation of the DNA-ends as depicted in FIG. 3.

[0057] More particularly FIG. 2 shows:

[0058] (A) A. vinelandii transformed with the plasmid pB5-Para-Cas9-PsacB-sgRNA-empty coding for Cas9 and a sgRNA without specific guide sequence. The transformants were plated on an agar plate containing Kanamycin.

[0059] (B) As in (A) but transformed with the plasmid pB5-Para-Cas9-PsacB-sgRNA-uppS5 that codes for Cas9 and sgRNA targeting the upp gene.

[0060] (C) As in (B) but transformed with the plasmid pB5-Para-dCas9-PsacB-sgRNA-uppS5 that codes for catalytically inactive Cas9 and sgRNA targeting the upp gene.

[0061] (D) As in (B) but transformed with the plasmid pB5-CLK_PsacB-sgRNA-uppS5 that encodes for Cas9-MtLigD-MtKu and sgRNA targeting upp gene.

[0062] The delivery of said plasmids into A. vinelandii was achieved by conjugation using E. coli S17-1.lamda.pir as donor cells.

[0063] A. vinelandii treated with pB5-CLK_PsacB-sgRNA-uppS5 were incubated on agar plates supplemented with 5-FU in order to select for upp mutants. Genomic DNA of a 5-FU resistant clone was isolated and the upp region was amplified by PCR. Results of Sanger sequencing showed the deletion of 308 bp (indicated in red in the sequence) region of the upp gene (FIG. 3).

Example 2

[0064] As shown in FIG. 4 the presence of Ku-LigD promotes the repair of DSB induced by Cas9 loaded with self-targeting sgRNA in P. putida.

[0065] More particularly FIG. 4 shows:

[0066] (A) P. putida transformed with the plasmid pB5-Para-Cas9-PsacB-sgRNA-uppS15 encoding Cas9 and sgRNA targeting the upp gene.

[0067] (B) As in (A) but transformed with the plasmid pB5-CLK_PsacB-sgRNA-uppS5 that encodes Cas9-MtLigD-MtKu and sgRNA targeting upp gene.

[0068] The delivery of said plasmids into P. putida was achieved by conjugation using E. coli S17-1.lamda.pir as donor cells.

Example 3

[0069] The presence of Ku and LigD from M. tuberculosis reduces the toxicity of self-targeting Cas9 nuclease in E. coli MG1655 (FIG. 5) and enables efficient introduction of NHEJmutations as shown in FIGS. 6 and 7.

[0070] More particularly FIG. 5 shows:

[0071] Chemically competent E. coli MG1655 was transformed either with pB5-Para-Cas9-PsacBsgRNA-bgaI or pB5-CLK_PsacB-sgRNA-bgaI. Both vectors encode wildtype Cas9 and a sgRNA targeting the lacZ gene. The vector pB5-CLK_PsacB-sgRNA-bgaI also expresses the proteins LigD and Ku from M. tuberculosis. The transformants were plated on selective agar plates and the numbers of colony forming units were determined.

[0072] FIG. 6 shows:

[0073] Chemically competent E. coli MG1655 were transformed either with pB5-Para-Cas9_PvegLigD_Ku or pB5-Para-Cas9_Pveg-LigD_PsacB_Ku. Both vectors encode wildtype Cas9, a sgRNA targeting the lacZ gene and express the proteins LigD and Ku from M. tuberculosis. Single colonies of the transformants were cultivated for preparation of electrocompetent cells.

[0074] After electroporation of the plasmid pUCP-PsacB-sgRNA-bgaI, containing lacZ-targeting sgRNA transcription unit, the transformants were plated on selective agar plates supplemented with ampicillin (100 .mu.g/ml), kanamycin (25 .mu.g/ml), 0.2% (w/v) arabinose and X-gal (80 .mu.g/ml). Frameshift mutations of lacZ-gene lead to white colored colonies.

[0075] FIG. 7 shows sequencing results of wildtype lacZ gene and five NHEJ-mutants obtained with Cas9 cleavage and subsequent repair by MtKu and MtLigD. The target site of Cas9 is shown in blue, the protospacer adjacent motif in red.

[0076] FIG. 1A shows the vector maps of pB5-Para-Cas9-PsacB-sgRNA, coding for the Cas9 protein and Psac-driven sgRNA, as used for the experiments with E. coli, P. putida and A. vinelandii.

[0077] FIG. 1B shows the vector maps of pB5-CLK_PsacB_sgRNA, coding for proteins Cas9, LigD and Ku, and Psac-driven sgRNA as used for the experiments with E. coli, P. putida, A. vinelandii.

[0078] FIG. 1C shows the vector maps of pB5-Para-Cas9_Pveg-LigD_Ku, as used for knockout of lacZ-gene in E. coli.

[0079] FIG. 1D shows the vector maps of pB5-Para-Cas9_Pveg-LigD_PsacB_Ku, as used for knock-out of lacZ-gene in E. coli.

[0080] FIG. 1E shows the vector maps of pUCP-PsacB-sgRNA-TrrnB, as used for knock-out of lacZ-gene in E. coli.

Sequence CWU 1

1

151102DNAStreptococcus pyogenes 1gcggatttcg tggatgggca gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 1022103RNAstreptococcus pyogenes 2gtcttggtgc tgatgtcggc agttttagag ctagaaatag caagttaaaa taaggctagt 60ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttt 1033102RNAStreptococcus pyogenes 3gaccatgatt acggattcac gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 1024105RNAstreptococcus pyogenes 4ggccagtgaa tccgtaatca gttttagagc tagaaatagc aagttaaaat aaggctagtc 60cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttt 10554107PRTStreptococcus pyogenes 5Ala Thr Gly Gly Ala Thr Ala Ala Gly Ala Ala Ala Thr Ala Cys Thr1 5 10 15Cys Ala Ala Thr Ala Gly Gly Cys Thr Thr Ala Gly Ala Thr Ala Thr 20 25 30Cys Gly Gly Cys Ala Cys Ala Ala Ala Thr Ala Gly Cys Gly Thr Cys 35 40 45Gly Gly Ala Thr Gly Gly Gly Cys Gly Gly Thr Gly Ala Thr Cys Ala 50 55 60Cys Thr Gly Ala Thr Gly Ala Ala Thr Ala Thr Ala Ala Gly Gly Thr65 70 75 80Thr Cys Cys Gly Thr Cys Thr Ala Ala Ala Ala Ala Gly Thr Thr Cys 85 90 95Ala Ala Gly Gly Thr Thr Cys Thr Gly Gly Gly Ala Ala Ala Thr Ala 100 105 110Cys Ala Gly Ala Cys Cys Gly Cys Cys Ala Cys Ala Gly Thr Ala Thr 115 120 125Cys Ala Ala Ala Ala Ala Ala Ala Ala Thr Cys Thr Thr Ala Thr Ala 130 135 140Gly Gly Gly Gly Cys Thr Cys Thr Thr Thr Thr Ala Thr Thr Thr Gly145 150 155 160Ala Cys Ala Gly Thr Gly Gly Ala Gly Ala Gly Ala Cys Ala Gly Cys 165 170 175Gly Gly Ala Ala Gly Cys Gly Ala Cys Thr Cys Gly Thr Cys Thr Cys 180 185 190Ala Ala Ala Cys Gly Gly Ala Cys Ala Gly Cys Thr Cys Gly Thr Ala 195 200 205Gly Ala Ala Gly Gly Thr Ala Thr Ala Cys Ala Cys Gly Thr Cys Gly 210 215 220Gly Ala Ala Gly Ala Ala Thr Cys Gly Thr Ala Thr Thr Thr Gly Thr225 230 235 240Thr Ala Thr Cys Thr Ala Cys Ala Gly Gly Ala Gly Ala Thr Thr Thr 245 250 255Thr Thr Thr Cys Ala Ala Ala Thr Gly Ala Gly Ala Thr Gly Gly Cys 260 265 270Gly Ala Ala Ala Gly Thr Ala Gly Ala Thr Gly Ala Thr Ala Gly Thr 275 280 285Thr Thr Cys Thr Thr Thr Cys Ala Thr Cys Gly Ala Cys Thr Thr Gly 290 295 300Ala Ala Gly Ala Gly Thr Cys Thr Thr Thr Thr Thr Thr Gly Gly Thr305 310 315 320Gly Gly Ala Ala Gly Ala Ala Gly Ala Cys Ala Ala Gly Ala Ala Gly 325 330 335Cys Ala Thr Gly Ala Ala Cys Gly Thr Cys Ala Thr Cys Cys Thr Ala 340 345 350Thr Thr Thr Thr Thr Gly Gly Ala Ala Ala Thr Ala Thr Ala Gly Thr 355 360 365Ala Gly Ala Thr Gly Ala Ala Gly Thr Thr Gly Cys Thr Thr Ala Thr 370 375 380Cys Ala Thr Gly Ala Gly Ala Ala Ala Thr Ala Thr Cys Cys Ala Ala385 390 395 400Cys Thr Ala Thr Cys Thr Ala Thr Cys Ala Thr Cys Thr Gly Cys Gly 405 410 415Ala Ala Ala Ala Ala Ala Ala Thr Thr Gly Gly Thr Ala Gly Ala Thr 420 425 430Thr Cys Thr Ala Cys Thr Gly Ala Thr Ala Ala Ala Gly Cys Gly Gly 435 440 445Ala Thr Thr Thr Gly Cys Gly Cys Thr Thr Ala Ala Thr Cys Thr Ala 450 455 460Thr Thr Thr Gly Gly Cys Cys Thr Thr Ala Gly Cys Gly Cys Ala Thr465 470 475 480Ala Thr Gly Ala Thr Thr Ala Ala Gly Thr Thr Thr Cys Gly Thr Gly 485 490 495Gly Thr Cys Ala Thr Thr Thr Thr Thr Thr Gly Ala Thr Thr Gly Ala 500 505 510Gly Gly Gly Ala Gly Ala Thr Thr Thr Ala Ala Ala Thr Cys Cys Thr 515 520 525Gly Ala Thr Ala Ala Thr Ala Gly Thr Gly Ala Thr Gly Thr Gly Gly 530 535 540Ala Cys Ala Ala Ala Cys Thr Ala Thr Thr Thr Ala Thr Cys Cys Ala545 550 555 560Gly Thr Thr Gly Gly Thr Ala Cys Ala Ala Ala Cys Cys Thr Ala Cys 565 570 575Ala Ala Thr Cys Ala Ala Thr Thr Ala Thr Thr Thr Gly Ala Ala Gly 580 585 590Ala Ala Ala Ala Cys Cys Cys Thr Ala Thr Thr Ala Ala Cys Gly Cys 595 600 605Ala Ala Gly Thr Gly Gly Ala Gly Thr Ala Gly Ala Thr Gly Cys Thr 610 615 620Ala Ala Ala Gly Cys Gly Ala Thr Thr Cys Thr Thr Thr Cys Thr Gly625 630 635 640Cys Ala Cys Gly Ala Thr Thr Gly Ala Gly Thr Ala Ala Ala Thr Cys 645 650 655Ala Ala Gly Ala Cys Gly Ala Thr Thr Ala Gly Ala Ala Ala Ala Thr 660 665 670Cys Thr Cys Ala Thr Thr Gly Cys Thr Cys Ala Gly Cys Thr Cys Cys 675 680 685Cys Cys Gly Gly Thr Gly Ala Gly Ala Ala Gly Ala Ala Ala Ala Ala 690 695 700Thr Gly Gly Cys Thr Thr Ala Thr Thr Thr Gly Gly Gly Ala Ala Thr705 710 715 720Cys Thr Cys Ala Thr Thr Gly Cys Thr Thr Thr Gly Thr Cys Ala Thr 725 730 735Thr Gly Gly Gly Thr Thr Thr Gly Ala Cys Cys Cys Cys Thr Ala Ala 740 745 750Thr Thr Thr Thr Ala Ala Ala Thr Cys Ala Ala Ala Thr Thr Thr Thr 755 760 765Gly Ala Thr Thr Thr Gly Gly Cys Ala Gly Ala Ala Gly Ala Thr Gly 770 775 780Cys Thr Ala Ala Ala Thr Thr Ala Cys Ala Gly Cys Thr Thr Thr Cys785 790 795 800Ala Ala Ala Ala Gly Ala Thr Ala Cys Thr Thr Ala Cys Gly Ala Thr 805 810 815Gly Ala Thr Gly Ala Thr Thr Thr Ala Gly Ala Thr Ala Ala Thr Thr 820 825 830Thr Ala Thr Thr Gly Gly Cys Gly Cys Ala Ala Ala Thr Thr Gly Gly 835 840 845Ala Gly Ala Thr Cys Ala Ala Thr Ala Thr Gly Cys Thr Gly Ala Thr 850 855 860Thr Thr Gly Thr Thr Thr Thr Thr Gly Gly Cys Ala Gly Cys Thr Ala865 870 875 880Ala Gly Ala Ala Thr Thr Thr Ala Thr Cys Ala Gly Ala Thr Gly Cys 885 890 895Thr Ala Thr Thr Thr Thr Ala Cys Thr Thr Thr Cys Ala Gly Ala Thr 900 905 910Ala Thr Cys Cys Thr Ala Ala Gly Ala Gly Thr Ala Ala Ala Thr Ala 915 920 925Cys Thr Gly Ala Ala Ala Thr Ala Ala Cys Thr Ala Ala Gly Gly Cys 930 935 940Thr Cys Cys Cys Cys Thr Ala Thr Cys Ala Gly Cys Thr Thr Cys Ala945 950 955 960Ala Thr Gly Ala Thr Thr Ala Ala Ala Cys Gly Cys Thr Ala Cys Gly 965 970 975Ala Thr Gly Ala Ala Cys Ala Thr Cys Ala Thr Cys Ala Ala Gly Ala 980 985 990Cys Thr Thr Gly Ala Cys Thr Cys Thr Thr Thr Thr Ala Ala Ala Ala 995 1000 1005Gly Cys Thr Thr Thr Ala Gly Thr Thr Cys Gly Ala Cys Ala Ala Cys 1010 1015 1020Ala Ala Cys Thr Thr Cys Cys Ala Gly Ala Ala Ala Ala Gly Thr Ala1025 1030 1035 1040Thr Ala Ala Ala Gly Ala Ala Ala Thr Cys Thr Thr Thr Thr Thr Thr 1045 1050 1055Gly Ala Thr Cys Ala Ala Thr Cys Ala Ala Ala Ala Ala Ala Cys Gly 1060 1065 1070Gly Ala Thr Ala Thr Gly Cys Ala Gly Gly Thr Thr Ala Thr Ala Thr 1075 1080 1085Thr Gly Ala Thr Gly Gly Gly Gly Gly Ala Gly Cys Thr Ala Gly Cys 1090 1095 1100Cys Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr Thr Thr Ala Thr Ala1105 1110 1115 1120Ala Ala Thr Thr Thr Ala Thr Cys Ala Ala Ala Cys Cys Ala Ala Thr 1125 1130 1135Thr Thr Thr Ala Gly Ala Ala Ala Ala Ala Ala Thr Gly Gly Ala Thr 1140 1145 1150Gly Gly Thr Ala Cys Thr Gly Ala Gly Gly Ala Ala Thr Thr Ala Thr 1155 1160 1165Thr Gly Gly Thr Gly Ala Ala Ala Cys Thr Ala Ala Ala Thr Cys Gly 1170 1175 1180Thr Gly Ala Ala Gly Ala Thr Thr Thr Gly Cys Thr Gly Cys Gly Cys1185 1190 1195 1200Ala Ala Gly Cys Ala Ala Cys Gly Gly Ala Cys Cys Thr Thr Thr Gly 1205 1210 1215Ala Cys Ala Ala Cys Gly Gly Cys Thr Cys Thr Ala Thr Thr Cys Cys 1220 1225 1230Cys Cys Ala Thr Cys Ala Ala Ala Thr Thr Cys Ala Cys Thr Thr Gly 1235 1240 1245Gly Gly Thr Gly Ala Gly Cys Thr Gly Cys Ala Thr Gly Cys Thr Ala 1250 1255 1260Thr Thr Thr Thr Gly Ala Gly Ala Ala Gly Ala Cys Ala Ala Gly Ala1265 1270 1275 1280Ala Gly Ala Cys Thr Thr Thr Thr Ala Thr Cys Cys Ala Thr Thr Thr 1285 1290 1295Thr Thr Ala Ala Ala Ala Gly Ala Cys Ala Ala Thr Cys Gly Thr Gly 1300 1305 1310Ala Gly Ala Ala Gly Ala Thr Thr Gly Ala Ala Ala Ala Ala Ala Thr 1315 1320 1325Cys Thr Thr Gly Ala Cys Thr Thr Thr Thr Cys Gly Ala Ala Thr Thr 1330 1335 1340Cys Cys Thr Thr Ala Thr Thr Ala Thr Gly Thr Thr Gly Gly Thr Cys1345 1350 1355 1360Cys Ala Thr Thr Gly Gly Cys Gly Cys Gly Thr Gly Gly Cys Ala Ala 1365 1370 1375Thr Ala Gly Thr Cys Gly Thr Thr Thr Thr Gly Cys Ala Thr Gly Gly 1380 1385 1390Ala Thr Gly Ala Cys Thr Cys Gly Gly Ala Ala Gly Thr Cys Thr Gly 1395 1400 1405Ala Ala Gly Ala Ala Ala Cys Ala Ala Thr Thr Ala Cys Cys Cys Cys 1410 1415 1420Ala Thr Gly Gly Ala Ala Thr Thr Thr Thr Gly Ala Ala Gly Ala Ala1425 1430 1435 1440Gly Thr Thr Gly Thr Cys Gly Ala Thr Ala Ala Ala Gly Gly Thr Gly 1445 1450 1455Cys Thr Thr Cys Ala Gly Cys Thr Cys Ala Ala Thr Cys Ala Thr Thr 1460 1465 1470Thr Ala Thr Thr Gly Ala Ala Cys Gly Cys Ala Thr Gly Ala Cys Ala 1475 1480 1485Ala Ala Cys Thr Thr Thr Gly Ala Thr Ala Ala Ala Ala Ala Thr Cys 1490 1495 1500Thr Thr Cys Cys Ala Ala Ala Thr Gly Ala Ala Ala Ala Ala Gly Thr1505 1510 1515 1520Ala Cys Thr Ala Cys Cys Ala Ala Ala Ala Cys Ala Thr Ala Gly Thr 1525 1530 1535Thr Thr Gly 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Ala Ala Thr Thr Gly Ala Thr Cys Gly Thr Ala3985 3990 3995 4000Ala Ala Cys Gly Ala Thr Ala Thr Ala Cys Gly Thr Cys Thr Ala Cys 4005 4010 4015Ala Ala Ala Ala Gly Ala Ala Gly Thr Thr Thr Thr Ala Gly Ala Thr 4020 4025 4030Gly Cys Cys Ala Cys Thr Cys Thr Thr Ala Thr Cys Cys Ala Thr Cys 4035 4040 4045Ala Ala Thr Cys Cys Ala Thr Cys Ala Cys Thr Gly Gly Thr Cys Thr 4050 4055 4060Thr Thr Ala Thr Gly Ala Ala Ala Cys Ala Cys Gly Cys Ala Thr Thr4065 4070 4075 4080Gly Ala Thr Thr Thr Gly Ala Gly Thr Cys Ala Gly Cys Thr Ala Gly 4085 4090 4095Gly Ala Gly Gly Thr Gly Ala Cys Thr Gly Ala 4100 410561368PRTStreptococcus pyogenes 6Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155 160Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310 315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550 555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu705 710

715 720His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790 795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys865 870 875 880Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1010 1015 1020Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser1025 1030 1035 1040Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1045 1050 1055Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1060 1065 1070Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1075 1080 1085Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1090 1095 1100Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile1105 1110 1115 1120Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1125 1130 1135Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1140 1145 1150Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1155 1160 1165Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1170 1175 1180Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys1185 1190 1195 1200Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1205 1210 1215Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1220 1225 1230Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1250 1255 1260Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val1265 1270 1275 1280Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1285 1290 1295His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1300 1305 1310Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1315 1320 1325Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1330 1335 1340Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile1345 1350 1355 1360Asp Leu Ser Gln Leu Gly Gly Asp 136572280PRTMycobacterium tuberculosis 7Ala Thr Gly Gly Gly Thr Thr Cys Gly Gly Cys Gly Thr Cys Gly Gly1 5 10 15Ala Gly Cys Ala Ala Cys Gly Gly Gly Thr Gly Ala Cys Gly Cys Thr 20 25 30Gly Ala Cys Cys Ala Ala Cys Gly Cys Cys Gly Ala Cys Ala Ala Gly 35 40 45Gly Thr Gly Cys Thr Cys Thr Ala Thr Cys Cys Cys Gly Cys Cys Ala 50 55 60Cys Cys Gly Gly Gly Ala Cys Cys Ala Cys Ala Ala Ala Gly Thr Cys65 70 75 80Cys Gly Ala Thr Ala Thr Cys Thr Thr Cys Gly Ala Cys Thr Ala Cys 85 90 95Thr Ala Cys Gly Cys Cys Gly Gly Thr Gly Thr Thr Gly Cys Cys Gly 100 105 110Ala Ala Gly Thr Cys Ala Thr Gly Cys Thr Cys Gly Gly Cys Cys Ala 115 120 125Cys Ala Thr Cys Gly Cys Gly Gly Gly Ala Cys Gly Gly Cys Cys Gly 130 135 140Gly Cys Gly Ala Cys Gly Cys Gly Cys Ala Ala Gly Cys Gly Cys Thr145 150 155 160Gly Gly Cys Cys Thr Ala Ala Cys Gly Gly Cys Gly Thr Cys Gly Ala 165 170 175Cys Cys 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Gly Gly Cys Thr Ala Gly Thr Cys Cys Gly Thr Thr Ala Thr225 230 235 240Cys Ala Ala Cys Thr Thr Gly Ala Ala Ala Ala Ala Gly Thr Gly Gly 245 250 255Cys Ala Cys Cys Gly Ala Gly Thr Cys Gly Gly Thr Gly Cys Thr Thr 260 265 270Thr Thr Thr Thr Thr Thr Thr Gly Cys Cys Thr Gly Gly Cys Gly Gly 275 280 285Cys Ala Gly Thr Ala Gly Cys Gly Cys Gly Gly Thr Gly Gly Thr Cys 290 295 300Cys Cys Ala Cys Cys Thr Gly Ala Cys Cys Cys Cys Ala Thr Gly Cys305 310 315 320Cys Gly Ala Ala Cys Thr Cys Ala Gly Ala Ala Gly Thr Gly Ala Ala 325 330 335Ala Cys Gly Cys Cys Gly Thr Ala Gly Cys Gly Cys Cys Gly Ala Thr 340 345 350Gly Gly Thr Ala Gly Thr Gly Thr Gly Gly Gly Gly Thr Cys Thr Cys 355 360 365Cys Cys Cys Ala Thr Gly Cys Gly Ala Gly Ala Gly Thr Ala Gly Gly 370 375 380Gly Ala Ala Cys Thr Gly Cys Cys Ala Gly Gly Cys Ala Thr Cys Ala385 390 395 400Ala Ala Thr Ala Ala Ala Ala Cys Gly Ala Ala Ala Gly Gly Cys Thr 405 410 415Cys Ala Gly Thr Cys Gly Ala Ala Ala Gly Ala Cys Thr Gly Gly Gly 420 425 430Cys Cys Thr Thr Thr Cys Gly Thr Thr Thr Thr Ala 435

44014445PRTMycobacterium tuberculosis 14Thr Cys Ala Gly Cys Cys Thr Gly Cys Ala Gly Cys Cys Cys Ala Thr1 5 10 15Gly Cys Ala Ala Cys Ala Gly Ala Ala Ala Cys Thr Ala Thr Ala Ala 20 25 30Ala Ala Ala Ala Thr Ala Cys Ala Gly Ala Gly Ala Ala Thr Gly Ala 35 40 45Ala Ala Ala Gly Ala Ala Ala Cys Ala Gly Ala Thr Ala Gly Ala Thr 50 55 60Thr Thr Thr Thr Thr Ala Gly Thr Thr Cys Thr Thr Thr Ala Gly Gly65 70 75 80Cys Cys Cys Gly Thr Ala Gly Thr Cys Thr Gly Cys Ala Ala Ala Thr 85 90 95Cys Cys Thr Thr Thr Thr Ala Thr Gly Ala Thr Thr Thr Thr Cys Thr 100 105 110Ala Thr Cys Ala Ala Ala Cys Ala Ala Ala Ala Gly Ala Gly Gly Ala 115 120 125Ala Ala Ala Thr Ala Gly Ala Cys Cys Ala Gly Thr Thr Gly Cys Ala 130 135 140Ala Thr Cys Cys Ala Ala Ala Cys Gly Ala Gly Ala Gly Thr Cys Thr145 150 155 160Ala Ala Thr Ala Gly Ala Ala Thr Gly Ala Gly Gly Thr Cys Gly Gly 165 170 175Cys Cys Ala Gly Thr Gly Ala Ala Thr Cys Cys Gly Thr Ala Ala Thr 180 185 190Cys Ala Gly Thr Thr Thr Thr Ala Gly Ala Gly Cys Thr Ala Gly Ala 195 200 205Ala Ala Thr Ala Gly Cys Ala Ala Gly Thr Thr Ala Ala Ala Ala Thr 210 215 220Ala Ala Gly Gly Cys Thr Ala Gly Thr Cys Cys Gly Thr Thr Ala Thr225 230 235 240Cys Ala Ala Cys Thr Thr Gly Ala Ala Ala Ala Ala Gly Thr Gly Gly 245 250 255Cys Ala Cys Cys Gly Ala Gly Thr Cys Gly Gly Thr Gly Cys Thr Thr 260 265 270Thr Thr Thr Thr Thr Thr Thr Gly Cys Cys Thr Gly Gly Cys Gly Gly 275 280 285Cys Ala Gly Thr Ala Gly Cys Gly Cys Gly Gly Thr Gly Gly Thr Cys 290 295 300Cys Cys Ala Cys Cys Thr Gly Ala Cys Cys Cys Cys Ala Thr Gly Cys305 310 315 320Cys Gly Ala Ala Cys Thr Cys Ala Gly Ala Ala Gly Thr Gly Ala Ala 325 330 335Ala Cys Gly Cys Cys Gly Thr Ala Gly Cys Gly Cys Cys Gly Ala Thr 340 345 350Gly Gly Thr Ala Gly Thr Gly Thr Gly Gly Gly Gly Thr Cys Thr Cys 355 360 365Cys Cys Cys Ala Thr Gly Cys Gly Ala Gly Ala Gly Thr Ala Gly Gly 370 375 380Gly Ala Ala Cys Thr Gly Cys Cys Ala Gly Gly Cys Ala Thr Cys Ala385 390 395 400Ala Ala Thr Ala Ala Ala Ala Cys Gly Ala Ala Ala Gly Gly Cys Thr 405 410 415Cys Ala Gly Thr Cys Gly Ala Ala Ala Gly Ala Cys Thr Gly Gly Gly 420 425 430Cys Cys Thr Thr Thr Cys Gly Thr Thr Thr Thr Ala Thr 435 440 44515480RNAEscherichia colideleted bases are underlined 15aagcgggaac gttacccagc gctcccgccg cgggcaagtc ccacggccac cgccggtagc 60ccacgtcact ctccggccgg ccgtgtttta attcaggcat cccatccccg agtgccggag 120ccctccatgc ccatccacga aatccgccat ccgctgatcc gccacaagct aggcctgatg 180cgccgtgccg acatcagcac caagagtttc cgcgaactgg cccaggaggt cggcgccctg 240ctcacttacg aggcgaccaa ggacctgccc ctggagaact accggatcga gggctggtgc 300ggcccggtgg aggtggagaa gatcgccggc aagaaaatca ccgtggtgcc catcctgcgc 360gccgggatcg gcatgctcga cggcgtgctg cgactgatcc cgaacgccaa ggtcagcgtg 420gtcggcctgt cgcgggacga ggaaaccctg gtcgcgcaca cctacgtgga aaagctggtg 480

Claims

1. A method for engineering and/or editing the genome of prokaryotes comprising the following steps: (i) providing a culture of prokaryotic cells, (ii) preparing a vector comprising an expression system encompassing at least one programmable DNA-binding and cleaving protein, and (iii) introducing said vector into said prokaryotic cells to target a specific DNA sequence in the genome of said prokaryotic cells.

2. A method for reconstituted DNA-end repair in a prokaryote comprising the following steps: (i) providing a culture of prokaryotic cells, (ii) preparing a vector comprising an expression cassette for a DNA-double strand break repair system comprising (a) at least one protein binding to the DNA-ends, and (b) at least one protein with DNA-ligase activity, and (iii) introducing said vector into said prokaryotic cells to enable introduction of double strand DNA breaks according to claim 1 in the genome of said prokaryotic cells.

3. A method for reconstituted DNA-end repair in a prokaryote comprising the following steps: (i) providing a culture of prokaryotic cells, (ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 50 nucleotides (nt) guide sequence of said sgRNA being complementary to desired stretches within the non-coding and/or putative regulatory regions upstream of the translation start codon of at least one gene of said prokaryotic cell, (iii) preparing a vector comprising an expression cassette comprising (a) at least one programmable DNA-binding and cleaving protein, (b) at least one optionally modified sgRNA, and (c) at least one DNA-end binding protein, and (iv) transforming said culture of prokaryotic cells with said vectors by standard methods (e.g. chemical transformation, electroporation, conjugation or transduction) to target the genome for the presence of a DNA sequence that is complementary to the 10 to 50 nt guide sequence of said sgRNA or protein-based nucleases like TAL- or Zn-finger proteins.

4. The method of claim 1, wherein the prokaryotic cells belong to bacteria.

5. The method of claim 1, wherein the vector is a plasmid or phage-DNA.

6. The method of claim 1, in which the vector is introduced into the prokaryotic cell by means of transformation, transduction or conjugation.

7. The method of claim 1, wherein the programmable DNA-binding and cleaving protein is selected from the group consisting of Zn-finger, TAL nucleases, meganucleases and RNA-dependent CRISPR-associated nucleases and mixtures thereof.

8. The method of claim 7, wherein the programmable DNA-binding and cleaving protein is selected from the group of CRISPR-Cas proteins belonging to class 2-type II CRISPR systems.

9. The method of claim 7, wherein the programmable DNA-binding and cleaving protein is Cas9 or Cpf1.

10. The method of claim 1, wherein the DNA-end repair protein is selected from proteins showing at least 30% identity in their primary sequence to protein Ku, and/or LigD of prokaryotes.

11. The method of claim 1, wherein the DNA-end repair protein is selected from proteins Ku and/or LigD encoded by Gram-positive bacteria.

12. The method of claim 1, wherein the DNA-end repair protein is proteins are selected from proteins Ku and/or LigD encoded by Mycobacteria.

13. The method of claim 1, wherein the DNA-end repair protein is selected from proteins Ku and/or LigD encoded by Mycobacterium tuberculosis.

14. An expression system, comprising (a) at least one Cas9, modified Cas9 or Cpf1 protein, (b) at least one optionally modified sgRNA or crRNA, and (c) protein Ku and/or LigD.

15. A vector comprising the expression claim 14.

16. A process for gene knock-out, deletion, replacement, editing, and/or genome wide knock-out screening in a prokaryote, comprising utilizing the method of claim 1.

17. A process for genome engineering and editing in prokaryotes, particularly targeted modification of a prokaryotic genome, such as disruption of gene function (knock-out), deletion of genomic loci or insertion of DNA elements in prokaryotes in combination with programmable nucleases that work via introduction of DNA-double strand breaks, comprising utilizing DNA-end binding and -repair proteins.